I. Field of the Invention
The field of the present invention relates to the transplantation of organs and tissues, and particularly to the conditioning of a xenograft organ while still in the donor to resist rejection antibodies in the graft recipient (accommodation). The invention also relates to methods for assessing accommodation within the donor animal.
II. Background
A major barrier to the transplantation of organs from one mammalian species to another is rejection of the xenografts. Much of the rejection is not related to tissue-specific antigens but results from the recipient being sensitized to the donor animal. For example, humans and Old World monkeys have circulating antibodies to the alpha galactosyl oligosaccharide expressed on tissues in other animals, including pigs. The antibodies bind to any transplanted pig xenograft, bind complement, and destroy the graft within an hour. This rapid reaction is referred to as hyper-acute rejection (HAR). The graft is rapidly destroyed by the binding of preformed natural antibodies to endothelial cells and fixation of the complement. Most of the preformed antibodies in humans and old world apes (>80%) are against Gal(alpha)1-3Gal epitopes (alphaGal).
Acute vascular xenograft rejection occurs at three to eight days post transplant. Induced and recurrent anti-donor antibodies bind to the endothelium, leading to endothelial activation, small vessel thrombosis, and recruitment of macrophages and NK cells. Acute xenograft rejection is also mediated by cellular rejection. In contrast to cellular allograft rejection, CD4+ cytotoxic lymphocytes contribute to the graft injury.
The current methods for prevention of HAR target the binding of antibody or the fixation of complement. Anti-donor antibodies or complement can be depleted from the blood of the recipient. Hyper-acute rejection was prevented in ABO mismatched cardiac allografts performed in baboons by infusion of soluble trisaccharides of the A and B antigen to neutralize the antibodies. Though the circulating antibodies persisted after discontinuing the oligosaccharides, some grafts showed prolonged survival. Cooper D. K., et al., Transplantation 56: 769-77 (1993). Transgenic pigs which express human complement inhibitors or with reduced expression of alphaGal have been created.
These technologies are useful on a short-term basis; however, they are not completely effective. Antibodies return rapidly after their removal and the procedure must be repeated frequently. Transgenic pigs are variable in the level of expression of the transgene. With both methodologies, episodes of HAR and acute vascular rejection are common. Efforts to suppress acute xenograft rejection using conventional chemotherapy have been only partially successful. In particular, the antibody response to pig xenografts has proved resistant to suppression.
To reliably achieve long-term survival of xenografts, immune tolerance or graft accommodation will be necessary. Immune tolerance involves programming the recipient's immune system to be specifically unresponsive to the graft. Accommodation refers to adaptation of the graft to be resistant to an existing immune response.
Partial immune tolerance to pig xenografts has been induced by ablating the recipient immune system and reconstituting it in the presence of porcine hematopoietic cells. Porcine hematopoietic cells are detectable a year later. This approach has three basic limitations. First, tolerance would not resolve the problem caused by pre-existing natural antibodies. Additional efforts, such as removal of pre-existing antibodies by immune adsorption would be required. Second, the recipient is subject to a prolonged period of immune deficiency, putting it at risk for opportunistic and zoonotic infections. Third, the tolerance would be to antigens expressed on the hematopoietic cells only. Tolerance would not be induced to the tissue-associated antigens. Pig heart and kidney xenografts were fulminantly rejected in baboons using this protocol.
The transplantation of pig thymi into immune ablated recipients enhances tolerance as the recipient pre-thymocytes mature in the porcine environment. Using this approach, porcine skin graft survival is markedly prolonged in mice and modestly prolonged in primates. The basic limitations described above with mixed chimerism would still be a problem.
Patent application No. PCT/US94/05844 teaches the induction of immune tolerance of recipient lymphocytes to xenografts by infusing lymphocytes into immune deficient donor animals. The tolerant cells are later harvested and transferred back to the recipient, conveying tolerance to the recipient. However, the preexisting immune response would limit the usefulness of that approach.
The mechanism of accommodation is unknown. It is not due to the depletion of antibodies or to replacement of donor endothelium with host endothelium within the graft. Immunohistochemistry of long term cardiac xenografts (hamster-to-rat) shows deposition of IgG, IgM, C3, and C6 on the endothelium, but minimal fibrin formation.
The possibility has been explored that accommodated endothelial cells have reduced expression of antigen. Though some reduction in antigens such as alpha gal was observed with accommodation, it was not thought to be sufficient to protect the graft. Parker W. et al., Am. J. Pathol. 152: 829-39 (1998).
It is known that accommodated grafts can be adoptively transplanted to a second recipient. The factors responsible for accommodation are present within the graft and do not require circulating regulatory cells or factors. Miyatake showed that if rejection of a hamster heart graft can be prevented in a rat recipient, the graft will also resist rejection when re-transplanted into a second recipient identical with the first recipient. T. Miyatake, N. Koyamada, W. W. Hancock, M. P. Soares, and F. H. Bach. Survival of accommodated cardiac xenografts upon re-transplantation into cyclosporine-treated recipients. Transplantation 65: 1563-1569, (1998). While the observation is of scientific value, it is not clinically useful. To apply this observation would require two identical subjects, such as human recipients, one who would host the donor organ until accommodation is achieved. The organ would then be procured and transplanted into the second subject. The very limited number of potential recipients with identical twins and the ethically unacceptable complications to the first recipient make this approach unfeasible.
Some success in achieving accommodation in cultured endothelial cells has been reported. Dorling A., et al., Xenotransplantation, 5: 84-92 (1998); and Dorling A., et al., Transplantation, 62: 1127-1136 (1996). Dorling et a. demonstrated that prolonged exposure in culture of porcine endothelial cells to normal human immunoglobulins produced endothelial cell changes suggestive of accommodation.
Apparent confirmation of these studies was provided by Shah et al., Fifth Congress of the International Xenotransplantation Association, Abstract 199 (1999). Minimal resistance to complement mediated cytotoxicity was achieved with 72 hours of culture. Better resistance was observed with 120 to 144 hours of incubation. On the other hand, others. were unable to confirm these studies using primary endothelial cell cultures. McKane W. et al. Fifth Congress of the International Xenotransplantation Association, Abstract 200 (1999). They suggested that the apparent resistance reported by others may be an artifact related to the use of immortalized endothelial cells, which constitutively express anti-apoptotic genes.
In vitro culture is unlikely to have significant clinical utility. Accommodated endothelial cells would not have significant utility by themselves. Furthermore, accommodation of cultured and transformed endothelial cells required a minimum of 72 hours and preferably 120 hours of culture. See Dorling et al. (1996), supra. If the observation were to be extended to ex vivo culture of whole organs maintained in culture, the organs would significantly deteriorate during this period.
Achieving accommodation within the recipient is very difficult, costly, and often ends in failure, with rejection of the graft.
Therefore, a need exists for a method of xenograft transplantation that avoids the high costs, the complications, and the high risk of failure associated with accommodation of the xenograft organ within the recipient after transplantation.